The human malaria parasite, Plasmodium falciparum, digests host hemoglobin as its amino acid source during the disease-causing, intraerythrocytic portion of the life cycle. Proteolytic degradation of hemoglobin and sequestration of the released toxic heme are accomplished within a parasite organelle known as the digestive vacuole (DV). The DV is the principal site of action for the endoperoxide-containing drug artemisinin and its derivatives. As multi-drug-resistant parasites continue to emerge, the artemisinin compounds are the most critical class of drugs for malaria control efforts over the next decade. Current research suggests artemisinin is activated to a highly reactive, alkylating free radical by ferrous heme iron within the parasite DV. However, the mechanism by which these reactive metabolites kill the malaria parasite is unknown. Several lines of evidence point to alkylation of DV proteins involved in hemoglobin digestion, oxidant defense or heme sequestration as the key mechanism of cell death. The goal of this proposal is to identify the targets of artemisinin toxicity while characterizing the proteome of the purified malaria parasite DV. State-of-the-art proteomic techniques, linked to the newly completed P. falciparum genome sequence data by mass spectrometry, provide an unprecedented opportunity to study parasite proteins as part of a functional system. The hypothesis is that artemisinin and its derivatives kill malaria parasites, in part, by interactions with proteins of the DV. Specific Aim 1 will use multidimensional protein identification technology (MudPIT) linked to the to P. falciparum genomic database to identify protein targets of artemisinin alkylation and toxicity in functional, purified DVs. This high-throughput approach will simultaneously identify the proteins comprising the DV proteome. In Specific Aim 2, drug-protein adducts from purified DVs and whole parasites exposed to [3H]-artemisinin will be identified using two-dimensional gel electrophoresis and mass spectrometry. In Specific Aim 3, changes in expression of proteins and corresponding RNA transcripts resulting from artemisinin exposure will be validated using real-time RT-PCR and western blotting. These studies will not only offer a more comprehensive understanding of the link between artemisinin toxicity and physiological functions of the DV, but also provide an important step in identifying new targets for antimalarial development.